1. Field of the Invention
This invention relates to novel magnetically responsive particles, useful as reagent carriers More particularly, the invention relates to magnetically responsive particles which can be coupled to or otherwise associated with, reactive groups, such as enzymes, haptens or antibodies, and used as carriers for analytical reagents and to the use thereof in performing assays. Also, a method is described for preparing the reagent carriers.
2. Brief Description of the Prior Art
A variety of magnetically responsive or attractable particles have been produced by several methods which incorporate magnetite, ferrite, chromium oxide or nickel powders into matrices to which reagents can be subsequently coupled
In one such method, a magnetically responsive powder is suspended in a solution of a polymer which is then gelled or precipitated to form a solid mass The solid so obtained is ground or milled to obtain an insoluble powder in which ferromagnetic granules are immobilized. See, for example, Robinson et al, Biotechnol Bioeng 15:603 (1973) and Pourfarzaneh et al, Methods of Biochemical Analysis, 28:281-3 (1982). The resulting particles are mostly oblong, jagged and of irregular size and shape. Thus, these particles exhibit poor flow properties. In addition, the number and mass of magnetic granules in individual particles are uncontrolled.
Another method involves the deposition of polymeric coatings on ferromagnetic powders by coacervation; for example, by the deposition and subsequent cross linking of albumin onto barium ferrite or magnetite See Ithakissios, et al, Clin Chem., 23:2072 (1977) and Widder et al, Clin Immunol and Immunopathol., 14:395-400 (1979) Other approaches to polymer-coated particles use polymerization, e.g., of acrylamide and/or methacrylamide with crosslinkers in the presence of magnetite, in a two phase (water/oil) system Also, magnetite particles themselves have been used as redox polymerization initiators. See Kronick et al, Science, 200:1074 (1978).
The traditional methods for producing such particles have relied on ferromagnetic powders or particles The particles generally exceed the critical size of a magnetic domain and, therefore, possess a substantial magnetic remanence Remanence is the magnetic induction that remains in a material after removal of the magnetizing force It is this property which causes such particles to remain aggregated, even in suspensions, after a magnetic field, to which they have been exposed, is removed Such particles are often spontaneously aggregated by the ambient terrestrial magnetic field. Hence, these particles are unsuited for use in automated assay systems which measure reaction rates, rather than end points, since such measurements require the periodic resuspension and redispersion of the particles.
To avoid problems associated with magnetic remanence, several methods have been developed which use superparamagnetic, rather than ferromagnetic materials, for the particles. Superparamagnetic materials, such as "ferrofluids" exhibit a very high magnetization at saturation (over 600 Gauss) and no magnetic remanence in the absence of an external magnetic field Examples include the microencapsulation of oil-based ferrofluids (see Kakimi et al, U.S Pat. No. 4,342,739) and the polymerization of aldehydes in the presence of aqueous ferrofluids See Rembaum et al, J. Macromolecular Science, Chemistry, A13:603-632 (1979); Rembaum, U.S. Pat. No 4,267,234; Larsson, et al, BiotechnoIogy Letters, 1 501-506 (1979); and Molday, et al, Nature, 268 437-438 (1977) However, due to their very small size, these particles exhibit a very slow magnetic response Further, the aqueous systems are susceptible to oxidation, so that these particles must be prepared fresh for each use or stored under an inert atmosphere.
Another such method "entraps"colloidal magnetite (a water-based ferrofluid) into or onto gel particles by prolonged contact with Sepharose beads. These particles leak iron oxide which prevents them from being useful in optical density or absorption measurements See Griffin & Mosbach, App. Biochem and Biotech., 6:283-292 (1981). These particles, therefore, are unstable, in that they do not retain permanently the iron oxide entrapped therein.
Another method has been described which provides a diluted array of superparamagnetic crystals in a glassy matrix See Mansfield et al, U.S. Pat. No. 4,297,337. These particles must be kept extremely small, e.g., less than one micron in diameter, to prevent their high specific gravity causing rapid sedimentation. The magnetic force on such small particles is very weak. Therefore, the fast magnetic responsiveness necessary for automated analytical systems cannot be achieved.
Thus, in summary, although magnetically responsive carrier particles have been prepared and used as reagent carriers in immunoassay procedures, each possessed characteristics which were undesirable for and compromised their usefulness in automated immunoassay systems.